Abstract Thirty percent of new HIV infections are transmitted by people unaware of their HIV status. Early detection of the presence of infectious levels of HIV virus could support public health efforts to stop the spread of the virus. The earliest biomarkers that can be used to diagnose HIV are elements of the HIV virion itself, including its RNA genome. In order to increase accessibility to early HIV testing, this project will develop the HIVAcute, a rapid self-test for the detection of HIV RNA based on isothermal RNA amplification that can be administered in the home. This device will allow collection of a finger-stick blood sample, gain power from any USB port, and deliver an automated diagnosis within 30 minutes. The work builds upon new developments in microfluidics and paper-based diagnostics, expanding the range of human samples that can be prepared and tested at the point of care. The long-term objective is to produce a commercial product for home testing. The specific aims are: 1. Develop and validate point-of-care-compatible sample acquisition and preparation strategies that rapidly deliver at least 25-125 copies of HIV RNA from a 50 L finger-stick blood sample in a form that supports iSDA- based detection. 2. Create and optimize isothermal real-time RT-iSDA assays for sensitive detection of HIV RNA and controls. 3. Design and build prototypes of the MD-NAAT platform that incorporate the output of Aims 1 and 2 for home HIV testing. 4. Develop near-product sample-to-result HIVAcute prototype and validate the prototype's user acceptability, ergonomics, and sensitivity as compared to laboratory standards. 5. Develop HIVAlert, a modification of HIVAcute that could use self-administered swabs to detect HIV+ semen in the vagina after unprotected sexual contact.